Document EP 0 511 108 describes broad spectrum yeast strains obtained by a program for crossing strains coupled with selection steps. The haploids and hybrid strains obtained by crossing them are selected on the basis of a high maltopermease activity and high maltase activity in the presence of glucose (and in the absence of maltose) and on the basis of a low invertase activity.
Document JP 9-149785 describes broad spectrum baker's yeast strains obtained by hybridization, wherein one of the haploid parent strains is deficient in invertase. The strains obtained are then selected on the basis of an invertase activity of 10 to 50 units and a maltase activity of at least 40 units, allowing said broad spectrum yeast strains to be obtained. According to that document, one unit of invertase activity corresponds to the formation of one milligram of reducing sugars per gram of yeast in one minute at 67% humidity.
Moreover, document EP 0 511 108 indicates that it is possible to reduce the invertase activity by means of molecular genetics by disrupting one or more SUC genes, preferably in haploids that have a maximum of one or two SUC genes.
The various selection steps, carried out on the basis of several criteria and involving at the same time the parent strains, the haploids and the hybrids, allow the chances of obtaining a broad spectrum yeast strain to be improved. However, the method for obtaining broad spectrum yeast strains as described in document EP 0 511 108 is relatively long and cumbersome in its execution.
Document EP 0 994 192 describes a novel excision cassette for use in yeast. This excision cassette means that no exogenous DNA is left in the yeast.
For example, document EP 0 994 192 describes the disruption of three copies of the SUC gene using, in succession, three excision cassettes each comprising: two recombinogenic sequences flanking two direct repeat sequences (mosaic sequence), themselves flanking a negative selection marker (RTA suicide gene) and a positive selection marker (kanamycin). The mosaic sequence is a non-heterologous DNA sequence from Saccharomyces cerevisiae comprising 97 base pairs.
The excision method described in document EP 0 994 192 is useful when disrupting a limited number of genes. However, it is not suitable for disrupting a larger number of genes: the excision method in fact involves constructing as many excision cassettes, and comprises as many disruption steps, as there are genes to be excised. The method described in EP 0 994 192 then becomes much too lengthy and cumbersome to carry out. In addition, the method described in document EP 0 994 192 cannot be used to disrupt more than 4 copies of the SUC gene, given the length of the recombinogenic sequence used and the size of the SUC gene.
The availability of broad spectrum yeast strains is useful to an industrial. In fact, it is then possible to produce, in the same unit and from a single yeast strain, yeasts intended for application to unsweetened dough and yeasts intended for application to sweet dough. Thus, the use of a broad spectrum yeast strain saves time and space in the factory, and therefore improves productivity.
The aim of the present invention is to provide a novel method that is rapid, effective and simple to implement in order to obtain novel broad spectrum baker's yeast strains.